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INTERACTIONS BETWEEN HOST and PHAGE ENCODED FACTORS SHAPE PHAGE INFECTION a Dissertation by DENISH KUMAR PIYA Submitted To

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INTERACTIONS BETWEEN HOST and PHAGE ENCODED FACTORS SHAPE PHAGE INFECTION a Dissertation by DENISH KUMAR PIYA Submitted To INTERACTIONS BETWEEN HOST AND PHAGE ENCODED FACTORS SHAPE PHAGE INFECTION A Dissertation by DENISH KUMAR PIYA Submitted to the Office of Graduate and Professional Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Chair of Committee, Ryland F. Young Co-Chair of Committee, Jason J. Gill Committee Members, Jennifer Herman Lanying Zeng Head of Department, Gregory D. Reinhart December 2018 Major Subject: Biochemistry Copyright 2018 Denish Piya ABSTRACT Evolution of phages and their bacterial hosts are directed by interaction between phage and host- encoded factors. These interactions have resulted in the development of several defense and counter-defense strategies such as DNA restriction and antirestriction systems. Type I restriction- modification (R-M) systems present a barrier to foreign DNA, including phage, entering the bacterial cell, by cleaving inappropriately modified DNA in a sequence-specific manner. Phages have evolved diverse mechanisms to overcome restriction systems. The temperate coliphage P1 encodes virion-associated proteins that protect its DNA from host type I R-M systems. By using genetic and biochemical analysis, it has been established that the P1 Dar (Dar for defense against restriction) system is comprised of at least six virion-associated proteins: DarB, Ulx, Hdf, DarA, DdrA and DdrB. DarB protects P1 DNA from EcoB and EcoK restriction in cis by an unknown mechanism and is incorporated into the virions only in the presence of Hdf, DarA and DdrA. Hdf and DarA have also been found to affect capsid morphogenesis, as their absence results in phage progeny with predominantly aberrant small heads. Examination of purified P1 proheads shows that Dar system proteins are incorporated into the virion before DNA packaging, and an N- terminal signal is required for DarB packaging. Twenty-four additional P1 genes of unknown function were disrupted and none were found to alter the antirestriction phenotype. A purification protocol for the ~250 kDa antirestriction protein DarB has been optimized, which will facilitate biochemical approaches for determining its mechanism of action. While the phage-host interactions of classical phages such as P1 are better understood, relatively little is known about how phages outside of the common paradigm organisms interact with their ii hosts. A high throughput genetic screen of the T1-like coliphage LL5 and the rV5-like coliphage LL12 was conducted against the E. coli Keio collection. This screen revealed host receptors required for both phages to initiate infection and two chaperones, PpiB and SecB, required for efficient propagation of phage LL5. iii DEDICATION I would like to dedicate this work to my late grandfather. He was my first teacher and taught me to read and write, and always encouraged me to pursue higher education. iv ACKNOWLEDGEMENTS I would like to thank my advisers, Dr. Jason J. Gill and Dr. Ry Young for believing in me and providing me with opportunity to work under their tutelage. Their support and words of wisdom have been very crucial in my journey through graduate school. The work presented in this dissertation would not have possible without their mentorship. Both of you have greatly inspired me to pursue science and excellence. I would also like to thank my committee members, Dr. Jennifer Herman and Dr. Lanying Zeng for their guidance, helpful discussion and comments throughout graduate school. I would like to thank past and present members of Gill Lab (Jackie Grimm, Season Xie, Abby Korn, Dr. Justin Leavitt, Justin Boeckman, Miguel F. Gonzales) for creating an awesome Lab environment and always being there for helpful discussions and assistance. I would also like to thank my undergraduate research mentees Brian Koehler, Judith Salazar, Leonardo Vara and Ashley Stonecipher for their help in conducting experiments and CPT staff Lauren Lessor for providing phages LL5 and LL12 used in this study. I also thank Dr. Matthew Taylor from Department of Animal Science at Texas A&M University for providing STEC strains used in this study. I also thank Dr. Junjie Zhang and Dr. Timothy Devarenne from Department of Biochemistry & Biophysics and Dr. Carlos F. Gonzalez from Department of Plant Pathology and Microbiology at Texas A&M University for allowing me to use their lab equipment. I also thank Dr. William Russell from Department of Chemistry at Texas A&M University (Currently at The University of Texas Medical Branch at Galveston) and Dr. Lawrence Dangott from Department of Biochemistry & Biophysics at Texas A&M University for assistance with mass spectrometry. v I would like to thank all CPT members and Supergroup members for their feedback and comments on data and presentations. Special thanks to Adriana C. Hernandez, Dr. Hem Thapa, Dr. Karthik Chamakura and Dr. Manoj Rajaure for teaching me several experimental techniques. I would also like to thank all administrative staffs of TAMU BioBio department for their assistance. Special mention to Mrs. Daisy Wilbert for always taking care of everything. I also thank my teachers, instructors and professors from Notre Dame School (Bandipur, Nepal), Small Heaven School (Narayanghat, Nepal), Budhanilkantha School (Kathmandu, Nepal) and Caldwell University (Caldwell, NJ, U.S.) for providing me with a solid foundation required for this journey. I would also like to thank all of my friends from my schools in Nepal and especially from Caldwell University and Texas A&M University for making life more fun and for being there at tough times. Finally, thanks for my parents for their encouragement and support throughout my educational journey. This journey has been only possible because their constant motivation since my first day at preliminary school. Thanks for my sisters as well for their support and love. Also, thanks to my wife for her patience and love and for sharing this journey with me. vi CONTRIBUTORS AND FUNDING SOURCES This work was supervised by a dissertation committee consisting of Dr. Ry Young (advisor), Dr. Jennifer Herman and Dr. Lanying Zeng of the Department of Biochemistry & Biophysics and Dr. Jason J. Gill (co-advisor) of the Department of Animal Science. All work for the dissertation was completed independently by the student. Graduate study was supported by start-up funding from Texas AgriLife Research and Texas A&M University to Dr. Jason J. Gill and from Deerland Enzymes. vii NOMENCLATURE E. coli Escherichia coli R-M Restriction-Modification viii TABLE OF CONTENTS Page ABSTRACT ....................................................................................................................................ii DEDICATION ...............................................................................................................................iv ACKNOWLEDGEMENTS ............................................................................................................v CONTRIBUTORS AND FUNDING SOURCES .........................................................................vii NOMENCLATURE ....................................................................................................................viii TABLE OF CONTENTS ...............................................................................................................ix LIST OF FIGURES ........................................................................................................................xi LIST OF TABLES .......................................................................................................................xiii CHAPTER I INTRODUCTION .....................................................................................................1 Bacteriophages ....................................................................................................................1 Phage P1 ..............................................................................................................................6 Host defense against phages ..............................................................................................13 Assembly of capsid-associated proteins ............................................................................31 Questions to be addressed ..................................................................................................33 CHAPTER II THE MULTICOMPONENT ANTIRESTRICTION SYSTEM OF PHAGE P1 IS LINKED TO CAPSID MORPHOGENESIS ...................................37 Introduction .......................................................................................................................37 Materials and methods .......................................................................................................42 Results and discussion .......................................................................................................52 Conclusions .......................................................................................................................77 CHAPTER III CHARACTERIZATION OF P1 GENES OF UNKNOWN FUNCTION AND PACKAGING OF DARB INTO P1 PROCAPSIDS .....................................83 Introduction .......................................................................................................................83 Materials and methods .......................................................................................................86 Results and discussion .....................................................................................................105
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